Reduced tar, nicotine, and carbon monoxide exposure while smoking ultralow- but not low-yield cigarettes.

An unresolved public health issue is whether some modern cigarettes are less hazardous than others and whether patients who cannot stop smoking should be advised to switch to lower-yield cigarettes. We studied "tar" (estimated by urine mutagenicity), nicotine, and carbon monoxide exposure in habitual smokers switched from their usual brand to high- (15 mg of tar), low- (5 mg of tar), or ultralow-yield (1 mg of tar) cigarettes. There were no differences in exposure comparing high- or low-yield cigarettes, but tar and nicotine exposures were reduced by 49% and 56%, respectively, and carbon monoxide exposure by 36% while smoking ultralow-yield cigarettes. Similarly, in 248 subjects smoking their self-selected brand, nicotine intake, estimated by blood concentrations of its metabolite cotinine, was 40% lower in those who smoked ultralow but no different in those smoking higher yields of cigarettes. Our data indicate that ultralow-yield cigarettes do deliver substantial doses of tar, nicotine, and carbon monoxide, but that exposures are considerably less than for other cigarettes.

Inhibition of microsomal lipid peroxidation by naphthoquinones: structure-activity relationships and possible mechanisms of action.

Menadione (2-methyl-1,4-naphthoquinone) is a remarkably potent inhibitor of microsomal lipid peroxidation, effective at submicromolar concentrations. Its possible mechanism of action and the relationship between naphthoquinone structure and antioxidant activity were the topics of this investigation. In the microsomal lipid-peroxidizing system dependent on NADPH and ferric pyrophosphate, menadione, at concentrations of 50 microM or higher virtually eliminated the accumulation of malondialdehyde and lipid hydroperoxides. In the NADPH-independent, cumene hydroperoxide-dependent system, menadione was also an effective antioxidant, but only in the presence of reducing equivalents. These and other observations indicate that a reduced form of menadione, either the hydroquinone or semiquinone, is the active antioxidant, and suggest that it may trap hydroperoxy radicals, alkoxy radicals, or other free radicals involved in propagating lipid peroxidation. Moreover, these results show that electron diversion per se cannot account for the antioxidant effects of menadione. A comparison of the antioxidant activities of eight 1,4-naphthoquinones indicated that methyl substitution of C-2, lack of steric hindrance at C-3 or C-5, and (in the case of weak acids) a relatively high pKa are favorable structural features associated with strong antioxidant activity.

Mutagenic and alkylating activities of organophosphate impurities of commercial malathion.

The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.

Metabolism of 2-aminoanthracene by BALB/c and C57BL mammary epithelium in vitro.

Aromatic primary amines, activated by P-450-dependent mixed-function oxidases have been shown to be mutagenic and carcinogenic in mammary gland of rodents. In this study, we compared 2-aminoanthracene (2AA) metabolism and inhibition in primary mammary gland cultures of BALB/c and C57BL mice. We sought to establish whether the rate or extent of metabolism was strain dependent as observed with polycyclic aromatic hydrocarbons even though the metabolic process would be different. Cells from either strain of mouse were uninjured by doses of 2AA up to 20 microM but produced polar metabolites of 2AA, some of which apparently formed covalent bonds with cellular macromolecules. In each case, covalent binding was inhibited by alpha-napthoflavone (alpha-NF), a mixed-function oxidase inhibitor. Any notable strain differences in 2AA metabolism and disposition was not observed. Results indicate that mouse mammary epithelial cells are capable of producing reactive metabolites of 2AA, probably via mixed-function oxidation. Thus, metabolic activation of 2AA in breast tissue may induce mutagenesis in mammary epithelium. The inhibitory effects of alpha-NF suggest that this compound may be of interest as a potential anti-carcinogen for the mammary gland.

Use of quinones in brain-tumor therapy: preliminary results of preclinical laboratory investigations.

Failure of current chemotherapeutic agents to effectively treat human brain tumors has prompted the search for alternative regimens based on the inherent metabolic pathways of target cells. One way to accomplish this goal would be to design drugs in an inactive form, which upon entry into the cell would be transformed to a toxic metabolite by a naturally occurring pathway. One such pathway may be the reductive activation of naphthoquinones with one or two side chains capable of alkylation, such as 2,3-dibromomethyl-1,4-naphthoquinone (DBNQ). This reductive activation can be catalyzed by the flavoprotein DT-diaphorase [NAD(P)H:quinone oxidoreductase]. We have found that both rat 9L and some human brain-tumor cell lines contain very high levels of this enzyme and that halogenated dimethyl naphthoquinones, such as DBNQ, are highly toxic to these cells in vitro. Moreover, we have found that the cytotoxic effects of DBNQ on human tumor and murine bone marrow stem cells can be prevented or lessened by pretreatment of the cells with dicoumarol, a potent inhibitor of DT-diaphorase. Since dicoumarol does not cross the blood-brain barrier, the potential exists for human brain tumors to be destroyed with halogenated dimethylquinones and for peripheral host toxicity to be prevented by coadministration of dicoumarol.

A methodology for the analysis of the preneoplastic antigen.

A highly sensitive assay for the epoxide hydrolase activity associated with the preneoplastic antigen (PNA) has been developed based on the synthesis of cis-stilbene oxide labeled with tritium at approximately 15 Ci/mmol. This assay allows the detection of elevated epoxide hydrolase activity in the serum of humans and rodents as well as in the culture medium bathing isolated hepatocytes. The integrity of the enzymatic assay was confirmed in rodents by precipitating the serum PNA activity using an antibody raised against the rat microsomal epoxide hydrolase. Methodology for the detection of PNA in serum will facilitate evaluation of this antigen as a marker for hepatic neoplasia in man and in experimental animals.

Inactivation of microsomal NADPH-cytochrome c reductase by sulfhydryl-reactive derivatives of menadione.

Five derivatives of menadione (2-methyl-1,4-naphthoquinone) having electronegative substituents on allylic carbons were prepared for study as sulfhydryl-reactive inactivators of mouse liver microsomal NADPH-cytochrome c reductase. Each of these naphthoquinones, incubated with dilute suspensions of microsomes, produced a loss of NADPH-cytochrome c reductase activity proportional to the initial naphthoquinone concentration. Each of the compounds also reacted with cysteine, as evidenced in the case of the halogenated compounds, by loss of reactive sulfhydryl groups and, in the case of 2-p-nitrophenoxymenadione, by the displacement of the leaving group, p-nitrophenol. Menadione, incubated under identical conditions, did not inactivate NADPH-cytochrome c reductase and was unreactive with cysteine. The requirement for a halogen or a nitrophenoxy substituent on at least one of the allylic carbons suggested that the mechanism of NADPH-cytochrome c reductase inactivation involves attack on critical microsomal nucleophiles, possibly sulfhydryl groups. The possible significance of these findings is discussed in relation to the antitumor activity and bioactivation of the halomethyl naphthoquinones.

Mutagenic impurities in 1,3-dichloropropene preparations.

A widely used pesticide, 1,3-dichloropropene [(DCP) CAS: 542-75-6], has been reported to be mutagenic to Salmonella typhimurium TA100, but large variations in specific mutagenic activity have been observed among different preparations. The purposes of this investigation were to determine the probable cause of the interpreparational variation and to provide new information on the nature of the mutagenic activity. Four preparations were assayed for mutagenic activity before and after silicic acid chromatography. None of the preparations retained mutagenic activity after chromatography, but each contained direct-acting mutagenic polar impurities. The specific mutagenic activities of the unpurified DCP samples appeared to be determined by the mutagenic activities of their polar impurities. A mixture of mutagenic polar impurities could be regenerated by refluxing a purified DCP preparation for 6 hours. The fraction of polar impurities from one of the preparations was analyzed by gas chromatography-mass spectroscopy. Although its composition was too complex to characterize completely, two known mutagens, epichlorhydrin (CAS: 106-89-8; 1-chloro-2,3-epoxypropane) and 1,3-dichloro-2-propanol (CAS: 96-23-1), were tentatively identified. In view of these results, future studies are required to establish whether DCP itself is a chemical carcinogen or whether its previously observed carcinogenicity resulted from the presence of mutagenic impurities.

Meperidine carboxylesterase in mouse and human livers.

Meperidine carboxylesterase activity was assayed in subcellular fractions of mouse and human liver by coupling the hydrolytic production of ethanol to the reduction of a tetrazolium dye. In mouse liver, the activity was found to be distributed among the mitochondrial, light mitochondrial, and microsomal fractions, whereas in human liver activity was found only in the microsomal fraction. The meperidine carboxylesterases in mouse liver and human liver were inhibited by two irreversible serine hydrolase inactivators (diisopropyl fluorophosphate and paraoxon) and by a reversible transition state analog (trifluoromercaptophenylacetone). Compared to the activities in mouse and human liver microsomes, the activity in mouse liver mitochondria was highly sensitive to the three inhibitors.

Possible role of DT-diaphorase in the bioactivation of antitumor quinones.

Menadione derivatives that are toxic to tumor cells are believed to be reduced intracellularly to species that react with DNA. In this communication, we report evidence that one of these derivatives, 3-bromomethylmenadione, is reduced by DT-diaphorases present in rat liver cytosol and in rat 9L brain tumor cells. Dicoumarol, an inhibitor of DT-diaphorases was found to inhibit both the reduction of 3-bromomethylmenadione and its mutagenicity to Salmonella typhimurium TA 97. Homogenates of rat 9L cells were found to contain relatively high levels of DT-diaphorase, suggesting that these tumor cells may be relatively sensitive to antitumor quinones that are activated by this enzyme.

Chemical characterization of direct-acting airborne mutagens: the functional group.

Durham NC air was sampled, extracted, and bioassayed for mutagenic activity in Salmonella typhimurium TA98. Portions of the extracts were treated with sodium borohydride over copper (II) acetylacetonate to reduce any nitroaromatic substances to their corresponding amines. All of the reduced extracts were not directly mutagenic, but the 2 that were derived from cold-weather air samplings did contain substances that could be activated oxidatively. These "indirect-acting" mutagens were present in the same 2 reduced extracts that contained detectable concentrations of aromatic amines. These results suggest that a major portion of the total mutagenic activity in air-pollution particles is contributed by nitro-substituted compounds that are detectable as their corresponding amines. They also suggest that the atmospheric concentrations of these substances may be high in the colder months of Durham's year.

Antimutagenic properties of liver homogenates, proteins and glutathione on diesel exhaust particulates.

Diesel exhaust particulates contain mutagens which are active in the Ames Salmonella typhimurium assay. The mutagens do not require liver enzymes for activation and, in fact, the addition of liver homogenates (S-9) to the Ames system decreases the mutagenicity of diesel exhaust samples. We have examined here the properties and components of S-9 that account for its antimutagenic effect. The antimutagenic effect of S-9 is not changed by heating S-9 in a boiling water bath for 5 min or by omission of the NADPH-generating system in the cofactor mixture. These experiments showed that the antimutagenic effect on S-9 is not enzymatic. The antimutagenicity of S-9 disappeared after the protein component of S-9 was removed by filtration. Exogenous albumin added to the Ames system mimicked the antimutagenic effect. Analysis of the albumin content of liver cytosol showed that 90% of the antimutagenic effect could be accounted for by the amount of albumin present. Glutathione added to diesel exhausts reduced mutagenicity, but the qualities of glutathione present in S-9 are too small to account for the antimutagenic effect of S-9. We conclude that the antimutagenic effect of S-9 is non-enzymatic and is most likely the result of non-specific protein binding of mutagens to liver albumin. The antimutagenic effect of glutathione on diesel exhausts suggests that the mutagens present are electrophiles.

Microsomal mixed-function oxidase and activities of some related enzymes in hyperplastic nodules induced by long-term griseofulvin administration in mouse liver.

Hepatic hyperplastic nodules induced in mice by long-term griseofulvin administration were examined for selected microsomal activities and responses to enzyme inducers. Despite a decrease in microsomal cytochrome P-450 in hyperplastic nodules, aminopyrine N-demethylase was at control levels. Benzopyrene hydroxylase activity was slightly lower in microsomes derived from hyperplastic nodules than in those of control liver. Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase was at control level, but reduced nicotinamide adenine dinucleotide- and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductases and the NADPH-ferricyanide reductase were increased. NADPH-supported lipid peroxidation was lower in microsomes from hyperplastic nodules than in those from control liver, whereas microsomal stearoyl coenzyme A desaturase activity was almost doubled in the nodules. NADPH-cytochrome c reductases isolated and semipurified from hyperplastic nodule and from control liver microsomes showed almost identical affinity for NADPH. Microsomal enzymes of hyperplastic nodules responded readily to phenobarbital induction, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis disclosed differences in the polypeptide patterns in the molecular weight range from 47,000 to 54,000 between microsomes derived from hyperplastic nodules and control livers.

Properties and inhibition of rat malathion carboxylesterases.

Two malathion carboxylesterase fractions, designated as esterase fraction A and esterase fraction B, that hydrolyze malathion were purified 13- and 18-fold, respectively, from rat liver microsomes. The two enzymes could not be distinguished kinetically, but fraction A contained at least one electrophoretic species not present in fraction B. The molecular weight of fraction A was estimated as 50,000-60,000; the molecular weight of fraction B was about twice this value. Incubation of [methoxy-14C]malathion with fraction A or fraction B resulted in a mixture of malathion alpha and beta monoacids, but the composition of the mixture produced by fraction A (alpha/beta = 1.5) differed from that produced by fraction B (alpha/beta = 0.2), indicating the presence of multiple species of carboxylesterases in mammalian liver microsomes. Isomalathion was substantially more potent as an inhibitor of both rat liver and rat serum malathion carboxylesterases than O,S,S-trimethyl phosphorodithioate. Isomalathion appeared to be equipotent in inhibiting the rat liver carboxylesterase-catalyzed reactions leading to either alpha or beta-monoacid. O,S,S-Trimethyl phosphorodithioate, on the other hand, preferentially diminished the reactions leading to alpha monoacid. In contrast, the rat serum carboxylesterase-catalyzed reactions leading to either alpha or beta monoacid were inhibited to approximately on equal degree by isomalathion and O,S,S-trimethyl phosphorodithioate.